The role of lineage in the temporospatial genesis of retinal bipolar cell subtypes

谱系在视网膜双极细胞亚型时空发生中的作用

• 批准号: 10571329
• 负责人: Ryan Delgado
• 金额: $ 12.36万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10571329
• 关键词: Back; Bar Codes; Benchmarking; Birth; Brain; CRISPR/Cas technology; Cell Survival; Cell division; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Competence; Complement; Complex; Cues; DNA; DNA Sequence; Data; Data Set; Development; Distant; Environment; Evolution; Eye; Foundations; Gene Expression; Generations; Genes; Genetic Transcription; Genomics; Goals; Guide RNA; Heritability; Individual; Inherited; Interneurons; Investigation; Knockout Mice; Laboratories; Light; Mentors; Modeling; Molecular; Morphology; Neurons; Output; Pattern; Phase; Physiological; Process; Production; Publishing; Retina; Retinal Cone; Retinal Ganglion Cells; Role; Series; Shapes; Sister; Site; Solid; Specific qualifier value; Supervision; Surface; Testing; Time; Tissues; Tracer; Training; Transgenic Organisms; Trees; Vertebrate Photoreceptors; Vision; Work; base; career; cell type; combinatorial; daughter cell; design; experience; in vivo; invention; medical schools; novel; postnatal; prime editor; progenitor; programs; response; retinal neuron; retinal progenitor cell; single-cell RNA sequencing; small molecule inhibitor; spatiotemporal; tool; tool development; training opportunity

PROJECT SUMMARY / ABSTRACT The retina is a cellularly complex tissue comprised of over 100 different cell types which work together to enable proprer vision. Bipolar cells are a diverse class of interneurons that connect rod and cone photoreceptors to retinal ganglion cells, the projection neurons of the retina, which then in turn send their output to the brain. There are currently 15 known subtypes of bipolar cells that differ in their connectivity, physiological responses to light, morphology, abundance, and gene expression profiles. A recent developmental study from our lab performed birthdating analysis to determine when different bipolar cell subtypes were born and found that bipolar cell subtype genesis was arranged into concentrated domains that changed over developmental time. When these domains were compared over time, they formed a wave-like temporospatial pattern of bipolar subtype genesis that spread over the entire extent of the retina. This result suggests a hierarchical model of bipolar subtype genesis in which early retinal progenitor cells generate a series of sub-lineages off-set in developmental time, that undergo a temporally ordered production of bipolar subtypes. To investigate this potential model, I have invented a novel molecular tool called SCRIBE (Sequential Combinatorial Recorder for Iterative Barcode Evolution) that functions as an “evolvable” lineage recorder. By iteratively adding barcode fragments to a genomic target site, SCRIBE aims to generate an evolving, heritable sequence that can be used to distinguish between sister sub-lineages across multiple levels of a clonal lineage tree. The goal of this proposal is to investagate how bipolar cell subtype specification is achieved in development. During the mentored portion of this proposal (K99), I will further develop SCRIBE for use in the in vivo retina and use it to investagate the temporal ordering of bipolar subtype production and the lineage relationships between postnatal RPCs that generate bipolar cells. In the independent portion of the proposal (R00), I will determine whether bipolar subtype specification is intrinsic or extrinsic, and investigate potential molecular regulators of bipolar subtypes specification. The completion of these aims will provide me with training in the fields of retinal development and cutting-edge molecular tool development, complementing my previous training experiences. I will perform the mentored portion of this proposal under the supervision of Dr. Constance Cepko who has been a leader in the fields of retinal development, lineage tracing, and in vivo molecular tool development. Furthermore, the broader scientific environment surrounding the Cepko lab at Harvard Medical School will provide outstanding training opportunities. Together, these experiences will provide me with a solid foundation that will support my long-term career goal of leading my own independent academic laboratory focused on studying the molecular bases of cellular diversity in the retina.

项目概要/摘要 视网膜是一种细胞复杂的组织，由 100 多种不同的细胞类型组成，这些细胞共同作用， 双极细胞是连接视杆细胞和视锥细胞的多种中间神经元。 视网膜神经节细胞（视网膜的投射神经元）的光感受器，然后依次发送它们的光 目前有 15 种已知的双极细胞亚型，它们的连接性不同， 对光、形态、丰度和基因表达谱的生理反应。 我们实验室的发育研究进行了出生日期分析，以确定不同的双极细胞何时 亚型诞生并发现双极细胞亚型起源被排列成集中的区域 随着时间的推移，这些领域发生了变化，它们形成了波浪状。 双极亚型起源的时空模式遍布整个视网膜。 提出了双极亚型起源的分层模型，其中早期视网膜祖细胞产生 一系列在发育时间中偏移的亚谱系，它们经历了双相情感障碍的按时间顺序产生 为了研究这种潜在的模型，我发明了一种名为 SCRIBE 的新型分子工具。 （用于迭代条形码进化的顺序组合记录器）充当“可进化”谱系 SCRIBE 旨在通过迭代地将条形码片段添加到基因组目标位点来生成一个记录器。 不断进化的、可遗传的序列，可用于区分多个级别的姐妹亚谱系 该提案的目标是研究双极细胞亚型规范的情况。 在本提案的指导部分 (K99) 中，我将进一步开发 SCRIBE。 在体内视网膜中的应用，并用它来研究双极亚型产生的时间顺序和 产生双极细胞的出生后 RPC 之间的谱系关系。 提案（R00），我将确定双相亚型规范是内在的还是外在的，并调查 这些目标的完成将为我提供双相亚型规范的潜在分子调节剂。 通过视网膜发育和尖端分子工具开发领域的培训，补充 我之前的培训经验将在以下人员的监督下执行本提案的指导部分。 Constance Cepko 博士是视网膜发育、谱系追踪和体内研究领域的领导者 此外，Cepko 实验室周围更广泛的科学环境。 哈佛医学院将提供出色的培训机会，这些经验将共同发挥作用。 为我提供坚实的基础，支持我实现独立的长期职业目标 学术实验室专注于研究视网膜细胞多样性的分子基础。